MICROSCOPY RESEARCH AND TECHNIQUE 20232-250 (1992)
Cytoskeletal Elements in Mammalian Spermiogenesis and Spermatozoa MARINA CAMATINI, ANITA COLOMBO, AND PATRIZZA BONFANTI Department of Biology, University of Milano, I-20133 Milan, Italy
KEY WORDS
Spermiogenesis, Spermatozoa, Mammals, Cytoskeleton
ABSTRACT Identification of the cytoskeletal elements and their role in the formation as well as the maintenance of head membrane compartmentalization is a much debated issue in mammalian spermatozoa. Data which have emerged during the last ten years are summarized. Those which have converged in a common opinion, such as the distribution of actin in mammalian spermiogenesis, are distinguished from those which have to be confirmed, such as the role of actin related proteins and actin in mature spermatozoa. SPERM MORPHOGENESIS The transformation of the spermatid into the spermatozoon is a complex process that proceeds through a series of cytological changes designated as “steps)’(Clermont and Leblond, 1955). These steps can be grouped into phases whose names are descriptive of the principal activities that characterize them. The first steps constitute the Golgi phase during which the Golgi apparatus begins the production of the acrosomal vesicle a t the side of the nucleus facing the lumen of the seminiferous tubule. This marks the anterior end of the future head of the sperm. At the opposite side of the spermatid, the centrioles begin to organize the tail fibers. During the cup phase, the acrosome spreads over the surface of the condensing nucleus; the spermatid rotates, so that the acrosomal complex faces the basement membrane of the seminiferous tubule and the developing tail faces the lumen. During the acrosome phase, the nucleus elongates and the cytoplasm is displaced, posteriorly, towards the growing flagellum, leaving, anteriorly, the acrosome covered only by the plasma membrane. An equatorial ring of fibrous material around the nucleus serves as the origin of an array of microtubules, the manchette, which extends as a cylinder into the distal cytoplasm. The manchette is involved in the displacement of the cytoplasm of the spermatid, and it disappears when the cytoplasm recedes from the nucleus. The final steps of spermatogenesis constitute the maturation phase. The nucleus becomes flattened and highly condensed, the centriole and columns of the connecting piece appear, and the mitochondria become organized around the proximal part of the flagellum to form the midpiece. Cytoskeletal Elements Involved in Sperm Morphogenesis It is a t present well known that cytoskeletal elements such as actin filaments (Masri et al., 1987; Vogl, 19891, microtubules (Gundersen et al., 1984; Vogl, 1988), and regulating proteins such as spectrin (Glenney and Glenney, 1983) and caImodulin (Camatini et al., 1986a; Weinman et al., 1986) are involved in cyto-
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plasm redistribution inside the cell, in cell shaping, and in morphogenetic movements of differentiating cells. The analysis of the events which regulate such phenomena may be performed by exploring the localization of cytoskeletal elements involved in cell shaping by the immunoelectron-microscopic technique. This technique employs antibodies against cytoskeletal proteins (Otey et al., 1986; Miller et al., 19871, which are characterized by immunoblots of one-dimensional (1D) or two-dimensional (2D) gel electrophoresis of the proteins under analysis. These antibodies are then used on fixed cells and processed for immunofluorescence, or on Lowicryl sections and cryosections and processed for immunoelectron microscopy. The antibodies can be recognized by specific anti-IgG conjugated with fluorescein or with colloidal gold particles (Carnatini et al., 1986a,c). Spermatogenesis is characterized by spectacular shape changes of both the nucleus and the cytoplasm. These changes are responsible for the final polarized form achieved in mature spermatozoa. Shape transformations are mediated by cytoskeletal elements, present inside the cell, which provide cytoplasmic tranlocation and organelle redistribution. This is the reason actin organization and the manchette of microtubules have been extensively analyzed in spermatogenic cells. While the role of microtubules is actually well defined, the presence of other cytoskeletal elements inside the spermatogenic cells has been long and controversially analyzed (Russell et al., 1986; Halenda et al., 1984, 1987). Numerous reports agree on the involvement of actin in sperm head morphogenesis (Vogl, 1989). Actin is certainly present in the spermatogenic cells themselves, and in attached Sertoli cell ectoplasmic specializations of all the mammalian species examined (see Camatini et al., 1987; Vogl, 1989; Fouquet et al., 1989).
Received April 24, 1990 accepted in revised form June 26,1990. Address reprint requests to Prof. Marina Camatini, Dipartmento di Biologia, Via Celoria 26, 1-20133 Milano, Italia.
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Fig. 1. Electrophoretic analysis (a) and immunoblot (b) of calmodulin (A) performed also in the presence of 1 mM Caf (B)or 5 m M EGTA (C). A a: PAGE profile (15%) of calmodulin from bovine testis. b Markers (lane l), polypeptides of boar sperm (lane Z), and purified calmodulin (lane 3) run in PAGE (15%), blotted, and stained with rabbit antiserum (lanes 4,5) and with anti-sheep antibodies (lanes 6, +
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7). B-C: SDS-PAGE (15% wiv) of markers (lane l), sperm extract (lane 21, and bovine testis calmodulin (lane 3) stained with 1% comassie brilliant blue; sperm extract (lane 4) and bovine testis calmodulin (lane 5) run in SDS-PAGE 15%, blotted onto nitrocellulose paper, and immunostained for calmodulin.
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Among these events, the movement of spermatocytes through the blood-testis barrier (Dym and Fawcett, 1970; Fawcett, 1975; Camatini et al., 1981; Pelletier, 1988), the translocation of spermatids to the luminal surface of the tubule, and the release of spermatozoa from the epithelium are the most significative ones. Although the underlying mechanism of these events is still unknown, the complex cytoskeletal network of Sertoli cells as well as the inter-Sertoli junctions are likely to be involved. Microfilaments (Russell et al., 1988; Vogl, 1989) and microtubules (Russell and Peterson, 1985) of Sertoli supporting cells during the development of spermatids and spermatozoa have been extensively studied, and their role is now well defined. Thus, sperm morphogenesis appears to be mediated by cytoskeletal elements in the supporting Sertoli cells as well as by cytoskeletal elements in specific regions of the shaping sperm heads.
Fig. 2. Fluorescent localization of calmodulin in boar (A-C) and rabbit (D-E) spermatozoa. A-B: Paraformaldehyde-fixed sperm. Fluorescence is on the acrosomal cap, the basal and middle piece of the tail. Bar = 5 pm. C: Methanol-fixed sperm. Fluorescence is present on the equatorial segment (arrowhead). Bar = 5 pm. D-E The subacrosomal bulges, the anterior part of the postacrosomal region, and the basal and middle piece of the tail are brightly fluorescent. F Phasecontrast image of E. Bar = 10 pm.
Role of Sertoli Cells in Sperm Morphogenesis Sertoli cells are thought t o be involved with many of the morphogenetic events that occur in the mammalian seminiferous epithelium during spermiogenesis.
ACTIN AND REGULATORY PROTEINS Among the cytoskeletal proteins, actin is the one that has received major attention during the past ten years (Pollard and Cooper, 1986). Owing to its importance in biology, it has been studied in considerable detail. First, it seemed that the discovery of the major class of actin-binding proteins could elucidate the assembly and function of the actin system in cells. Second, it was apparent that more complicated mechanisms must be involved. Although most of the regulatory proteins have been localized in cells, there is almost no direct experimental evidence regarding the function of such regulatory proteins in living cells. There are a t least four different families of proteins that bind primarily to actin monomers (Pollard and Cooper, 1986) and every one of the actin monomer binding proteins studied to date forms a complex with actin that does not polymerize, as free actin monomers do. This inhibition is explained by the ability of these proteins to sequester monomers in a nonpolymerizable complex. Comparison of the primary structure of actin shows that this protein has been highly conserved during evolution (Korn, 1982). There are less than 5% differences in the amino acid sequence of muscle and cytoplasmic actins from protozoa to vertebrates; plants have multiple actins that differ from each other and from animal actins by more than 10%in the amino acid sequence. Based on the amino acid sequence homologies, all Of invertebrates appear to be direct descendants of the cytoplasmic actins found in protozoa and fungi, while the muscle actins of vertebrates form an easily distinguishable family. Despite these differences in actin isoformsexpressed, it has long been clear that actin plays a central role in motility beyond its classic contractile function in muscle. Membrane-Skeleton in Non-Muscle Cells The role of actin in non-muscle cells remains an intriguing puzzle. Recent findings and new approaches (Bennett, 1985; Marchesi, 1985; Mercier et al., 1989) have elucidated the composition of a membrane-bound
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Fig. 3. Sections of boar (A-C)and rabbit (D) sperm heads immunostained for calmodulin, detected with antibody conjugated to gold particles. These are distributed inside the acrosome (A), along the equatorial segment (E)and the postacrosomal region (PA). Arrowheads evidence the stain along the E segment (B). A: Bar = 0.45 pm.
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B: Bar = 0.15 pm. The head of a late spermatid shows gold particles inside the acrosome and along the microfilaments (arrows) of the surrounding Sertoli cell (C). In this longitudinal section (D) the particles are present on the postacrosomal region (PA) and the subacrosomal bulges. E, equatorial segment; N, nucleus. C-D: Bar = 0.29 pm.
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Fig. 4. Specificity of anti-actin antibodies on different actins and boar sperm extracts. Amido black staining (A) and immunostaining (€3-C) of boar spermatozoa (lanes 1) and purified muscle actin (from rabbit skeletal muscle) (lanes 2), purified smooth-muscle actin (from
chicken gizzard) (lanes 3), and purified non-muscle actin (from Pfiysarum) (lane 41, blotted to nitrocellulose. A Polypeptides run on 10% SDS-PAGE. B: Immunostaining with the anti-y antibody. C: Immunostaining with the anti-a antibody.
lattice. Among these studies, the cytoskeletal network, underlying the erythrocyte membrane a t the molecular level, has received considerable attention, and the model of the membrane-skeleton, which gives the erythrocyte its stability and unique viscoelastic properties, has been well defined (Bennett, 1985; Marchesi, 1985). In search of the supramolecular organization of the erythrocyte membrane-skeleton, extensive studies have been directed a t the structure and chemical nature of spectrin, including its binding with other proteins, such as actin, ankyrin, and band 4.1 (Morrow, 1989).The erythrocyte membranes have played a leading role as a model system in research on the attachment of the cytoskeleton to the membrane. However, the applicability of this system to other cell types remains a matter for speculation. The hypothetical model of the membrane-skeleton of the nicotinic acetylcholine receptor (Bloch and Morrow, 1989) involves oligomeric actin which cross-links spectrin. Thus, it is apparent that these two proteins are gaining attention when receptor organization, topographic membrane assembly, and membrane trafficking appear to be functions expressed in non-erythroid cells.
al., 1981; Primakoff and Myles, 1983; O’Rand et al., 1985; O’Rand and Fisher, 1987). It has been reported that plasma membrane proteins in the postacrosomal (PA) region exibit little lateral mobility in the rabbit mature spermatozoa in contrast to the lateral mobility of proteins in the plasma membrane of the acrosomal region (Welch and O’Rand, 1985). Identical observations have been made in guinea pig (Primakoff and Myles, 1983) and rats (Gaunt et al., 1983). These results imply an anchoring mechanism below the plasma membrane in particular head regions. If this assumption is correct, the colocalization in gametes of spectrin and calmodulin (Virtanen et al., 1984; Sobel et al., 1988; Camatini et al., 1991), of actin and calmodulin (Camatini and Casale, 19871, and of spectrin and actin (Reima and Lehtonen, 1985) suggests that they might have a cytoskeletal-membrane role. Besides these regulatory proteins, other cytoskeletal elements have been described in mammalian spermatozoa. Intermediate filament proteins, such as vimentin (Virtanen et al., 1984; Ochs et al., 1986; Olson et al., 1987) and keratin (Ochs et al., 1986), were found in human spermatozoa. Moreover, Longo et al. (1987) have described a novel class of cytoskeletal proteins which surround the nuclei of bull and rat spermatozoa.
Membrane-Cytoskeletal Elements in Sperm Heads The mammalian sperm head represents a fascinating system for the compartmentalization of its regions, performed by the plasma membrane (PM), the outer acrosomal membrane (OAM), and the inner acrosomal membrane (IAM). Protein mobility appears to be restricted in particular areas of these membranes, which are maintained after the acrosome reaction (Myles et
IDENTIFICATION OF CYTOSKELETAL ELEMENTS IN SPERMIOGENESIS AND SPERMATOZOA During the last ten years, numerous laboratories have directed their research at the analysis of cytoskeleta1 elements involved in the compartmentalization of
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Fig. 5. Two-dimensionalgels (A,B) and immunoblots(C,D) of rabbit sperm extracts. A: 2D electrophoresis of sperm extract. B: Comigration of sperm extract and chicken gizzard actin. The presumptive y-actin spot (white arrowhead) and p-actin spot (black arrowhead)
are circled anti-y (C) and anti-a (D)antibodies show one spot each. The cathode (-1, the anode (+), and pH range (6.5-4.5) are indicated. Arrow: 45,000 molecular weight.
the head membranes of mammalian spermatozoa with different immunocytochemical techniques. The distribution of several cytoskeletal proteins in mammalian spermiogenesis and spermatozoa have been described in numerous species (see Olson et al., 1987). Actin has received considerable attention, and its presence during spermiogenesis is well documented (see Camatini et al., 1986b, 1987; Vogl, 19891, since a consistent distribution pattern has emerged. Despite uniform results obtained with various techniques, it is reported that actin is not maintained in all mature spermatozoa of the species examined. Moreover, it appears that actin is not placed in the same cell compartments. Thus, a common functional role may be suggested for actin during spermiogenesis, while it remains a question for mature spermatozoa.
Among proteins directly or indirectly associated with actin, calmodulin and spectrin are the ones examined by several laboratories.
Calmodulin Calmodulin was first demonstrated in the soluble fraction of mammalian sperm (Jones et al., 1978) and recently it has been found associated with the PM proteins of bovine epididymal spermatozoa both in the presence of EGTA and C a i + (Noland et al., 1985; Peterson et al., 1989). Moreover, it has been suggested (Olson et al., 1985) that calmodulin may be bound to the OAM complex and is released during Ca+ influx. These biochemical findings demonstrate the presence of multiple classes of calmodulin binding proteins in mammalian sperm as well as in other cells (Van Eldik +
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Fig. 6. Localization of actin on early boar spermatids. Testis Lowicryl sections are stained with an anti-actin monoclonal antibody, recognized with goat anti-mouse 10 nm gold particles. Actin (arrowheads) is visible on the subacrosomal area (A-D) and on the microfilaments (arrow) of Sertoli cell (S) specializations (B-D). A, acrosome; N, nucleus. A: Bar = 0.71 km. B: Bar = 0.29.C: Bar = 0.35 pm. D: Bar = 0.40 pm.
Fig. 7. Localization of actin on early rabbit spermatids. Testis Lowicryl sections are stained with the anti-cx actin antibody, evidenced with goat anti-rabbit 10 nm (A-C) and the monoclonal antiactin antibody, evidenced with goat anti-mouse 5 nm (D.) Actin distribution is visible in the subacrosomal area (A-C, arrowheads). The particles are continuous in the thin space between the acrosome and
the nucleus (AX). When the spermatid head flattens (D), particle density in the subacrosomal area is lower. A, acrosome; N, nucleus. Arrows indicate the final region of contact between the acrosomal vesicle and the nucleus. A: Bar = 0.9 km. B: Bar = 0.4 Fm. C: Bar = 0.38 pm. D: Bar = 0.26 pm.
Fig. 8. Localization of actin on boar late spermatids (arrowheads) and on Sertoli cell specializations (arrows). N, nucleus. In longitudinal sections (A) spermatid heads present discontinuous particles under the IAM (arrowheads). The label, which caps the heads, corresponds to Sertoli (S) microfilaments. A Sertoli-Sertoli junctional specialization (double arrow) is indicated. A Bar = 0.90 pm. Spermatid cross-sections a t the acrosome level (B,C,E) and a t the PA re-
gion (D). B Bar = 0.24 pm. C:Bar = 0.26 pm. D: Bar = 0.50 pm. E: Bar = 0.23 km. The particles are present under the plasma membrane in the postacrosomal region of a mature spermatid (D). In a longitudinal tangential section (F),the mature spermatid is enwrapped by Sertoli specializations, marked with gold particles. F: Bar = 0.33 pm.
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Fig. 9. Localization of actin in rabbit late spermatids (A-E).Actin immunostaining is performed with the monoclonal anti-actin antibody recognized by goat anti-mouse 5 nm (C,D) or goat anti-mouse 10 nm (A) and anti-ol antibody is recognized by goat anti-rabbit 10 nm (B,D). A, acrosome; N, nucleus; PA, postacrosomal region. A Bar = 0.25 pm. B: Bar = 0.3 km. C: Bar = 0.18 km. Arrowheads indicate
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the staining on the subacrosomal and postacrosomal region; arrows indicate the Sertoli cell (S)specialization. Discontinuous particles are present in the subacrosomal area (A,B,D). The postacrosomal region is marked by particles close to the plasma membrane (0. The label on surrounding microfilaments parallels the membrane of the spermatids (D,E). D: Bar = 0.29 pm. E: Bar = 0.45 *m.
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and rabbit (Fig. 2D,E) spermatozoa. Immunoelectron microscopy furnished additional details (Weinman et al., 1986; Camatini et al., 1986a) and calmodulin appears to be localized along the postacrosomal membrane (PA) and the cytoplasmic face of the OAM of the equatorial (E) segment in boar (Fig. 3A,B) and rabbit (Fig. 3D) spermatozoa. Moreover, calmodulin had been localized in boar (Fig. 3C) and bull (Weinman et al., 1986) spermatids. Rabbit spermatozoa, which have four subacrosomal bulges (Phillips, 1972), have calmodulin also in these regions (Fig. 3D). The distribution of calmodulin in mammalian spermatozoa supports the idea that this protein is related to the acrosome reaction, playing a key role in the fertilization process.
Fig. 10. Immunofluorescence localization of actin on boar (A-B) and rabbit (D) spermatozoa. Fluorescence is present at the curved region of the heads, at the equatorial segment, and at the pericentriolar area (A). Sperm, which have lost their acrosomes,present a weak fluorescent line at the IAM (arrows) (B). The absence of label on sperm stained with rhodamine-phalloidin is evident (C). Fluorescent (D) and corresponding phase-contrast(E) images of rabbit sperm. The postacrosomal regions (D) and the subacrosomal bulges are brightly fluorescent (arrow). A-E: Bar = 10 pm.
and Burgess, 1983; Hopper and Kelly, 1984). The specificity of the anti-calmodulin antibodies we used was tested by immunostaining blots of sperm extracts and purified calmodulin (Fig. 1A); the presence of a single band in the 17 kDa region confirms the reactivity of the antibody. Thus, biochemical results demonstrate the existence of calmodulin and its different electrophoretic mobility in the presence of Ca ' (Fig. 1B) or EGTA (Fig. 1 0 . An indirect immunofluorescence technique showed calmodulin in ejaculated guinea pig (Jones et al., 1980), bull (Feinberg et al., 19811, boar (Figs. 2A-0, +
Actin Actin in ejaculated mammalian spermatozoa has been studied by numerous authors (Talbot and Kleve, 1978; Clarke and Yanagimachi, 1978; Tamblyn, 1980; Clarke et al., 1982; Flaherty et al., 1983, 1986, 1988; Virtanen et al., 1984; Welch and ORand, 1985; Ochs and Wolf, 1985; Camatini et al., 1986b,c; 1987; Camatini and Casale, 1987; Casale et al., 1988; Peterson et al., 1990). The difficulties encountered in its localization, using antibodies of different specificities with immunofluorescence, with immunoelectron microscopy, and with biochemical methods, are certainly related to the small amount of actin in these cells. Comparing the results obtained with anti-actin antibodies directed against actins of various origin (Fig. 4A-C), we have demonstrated that the differences in antibody reactivity depend on the relative proportion of the a,(3, and y isoforms present in the actins examined. Actin was extracted from human spermatozoa (Ochs and Wolf, 19851, extracted and identified on 2D PAGE immunoblots as a single spot of PI = 5.45 in rabbit spermatozoa (Welch and ORand, 1985). We have obtained 2D gel separation of actin in boar (Casale et al., 1988) and rabbit spermatozoa (Fig. 5A,B), and we have identified in immunoblots of these gels two spots (Fig. 5C,D) corresponding to y and p isoforms of actin. Thus, we can assume that rabbit and boar spermatozoa express the P and y isoforms usually present in nonmuscle cells. The morphological results have shown that actin is present from the early cup phase of spermiogenesis in ground squirrel (Vogl et al., 19861, rat (Russell et al., 1986; Vogl et al., 19861, guinea pig (Halenda et al., 19871, boar (Fig. 6A-D) and rabbit (Fig. 7A-D). The shaping of the acrosome around the anterior pole of the nucleus apparently follows an identical scheme in all mammals studied so far. Actin becomes apparent when the acrosomal vesicle makes contact with the anterior pole of the nucleus. As has been shown with different immunostaining techniques, the restricted area of juxtaposition between the flattening membrane and the nuclear envelope is always positive to the stain. Immunoelectron microscopy shows that the restricted area of juxtaposition between the flattening membrane and the nuclear envelope is labelled by anti-actin antibody. This label parallels the shaping of the vesicle over the nucleus (Figs. 6, 7, A,B). Apparently, the inner acroso-
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Fig. 11. Ejaculated boar sperm stained with a monoclonal antiactin antibody evidencedwith goat anti-mouse 10 nm. A, acrosome;E, equatorial segment; PA, postacrosomal region. The longitudinal tangential section of the sperm head (A) shows gold particles on the E
segment. A: Bar = 0.71 pm. Particles are aligned between the OAM of the equatorial segment and the PM, in a cross-sectionedhead. B: Bar = 0.29 km. The sagittally sectioned head (C) is labeled only at the equatorial segment. C: Bar = 0.38 km.
ma1 membrane and the nuclear envelope are connected by actin for the entire duration of their contact. Then chromatin condenses and the spermatid head flattens (Fig. 8A-F). The thin space, separating the acrosomal vesicle from the nucleus, maintains the same thickness until the spermatids mature, even though it is less marked by gold particles (Figs. 8B-E, 9B,D). This observation might account for a changed distribution of actin in late spermatids, or for a complete loss of it (Halenda et al., 1987). Actin filaments in Sertoli cell specializations become more evident in coincidence with spermatid elongation, since ectoplasmic junctional areas expand to surround all the spermatid head (Figs. 8C-F, 9B,D,E). Actin is maintained in correspondence of the equatorial segment and the postacrosomal region after ejaculation in human (Clarke et al., 1982; Virtanen et al.,
1984; Ochs and Wolf, 19851, boar (Tamblyn, 1980; Camatini et al., 1987; Peterson et al., 1990), bull, hamster (Flaherty et al., 1988), and rabbit (Welch and ORand, 1985; Camatini et al., 1987) spermatozoa. However, its conservation in ejaculated spermatozoa does not seem to be the rule. In some cases it is lost during maturation, as reported for rat late spermatids (Russell et al., 19861, for plains mouse epididymal spermatozoa (Flaherty et al., 1986), and for guinea pig mature sperm (Halenda et al., 1987). Immunofluorescence has been the technique most used in actin detection, even though it cannot account for a precise localization (Fig. 10A-D). The immunoelectron microscopic technique has furnished details on the distribution of this protein in boar (Fig. 11A-C) and rabbit (Fig. 12A-D) spermatozoa. Actin appears to be conserved in particular head membrane regions, which will be maintained after the
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Fig. 12. Actin localization on rabbit ejaculated sperm (A-D). Actin is detected with the anti-a antibody recognized with goat antirabbit 10 nm in A and it is detected with the monoclonal antibody recognized with goat anti-mouse 15 nm in B and C. Arrows, gold particles; E, equatorial segment; arrowheads, subacrosomal bulges;
PA, postacrosomal region. Longitudinal sections show particles on the subacrosomal bulges (A,B,D) and on the anterior part of the postacrosomal region. A: Bar = 0.31 km. B: Bar = 0.33 km. D: Bar = 0.15 wm. The anterior part of t h e postacrosomal region presents clusters of particles. C: Bar = 0.23 Fm.
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Fig. 15. Fluorescent localization of anti-chicken erythrocyte spectrin (A)and anti-a-mouse brain fodrin (B) antibodies. Fluorescence is on the acrosomal cap, the subacrosomal bulges, and the principal piece of the tail.
Vogl et al., 1986),rhodamine-phalloidin stain in rabbit spermiogenesis (Welch and O'Rand, 1985), and protofilaments associated with boar membrane spermatozoa (Peterson et al., 1990). In the species we examined (Camatini et al., 1986b; 1987), actin is present in oligomeric form and in correspondence with membrane regions, whose domains must be anchored. Fig. 13. SDS electrophoretic separation on 7.5% slab gel of high molecular weight markers (M),brain extract (lane l), sperm extract (lane 2), chicken erythrocyte spectrin (lane 3), and sperm extract run with purified spectrin (lane 4). Numbers indicate the protein marker molecular weight in kDa.
Spectrin Evidence has emerged recently on the presence of a spectrin-like protein in gametes, zygotes, and embryos of sea urchins and mice (Schatten et al., 1986; Lakoski Fig. 14. Blots of electrophoretically separated proteins from brain et al., 1989), in ejaculated human (Virtanen et al., (lane 1)and sperm (lane 2) extracts probed with different antibodies. 1984) and rabbit (Camatini and Casale, 1988; CamaA Anti-chicken spectrin. B: Anti-mouse brain a-fodrin. C:Anti-calf lens p230 spectrin. D Detection of calmodulin binding proteins on tini et al., 1991) spermatozoa, and in mouse gametogesperm polypeptides, after electrophoresis and transfer onto nitrocel- nesis (Damjanov et al., 1986).While the role of spectrin lulose paper. The blots were incubated in the presence of either 1mM and related proteins in forming the membrane-skelCaC12 (lane 1)or 5 mM EGTA (lane 2). eton in the erythrocytes is well defined, in non-erythroid cells it is less clear. Spectrin seems to be unnecessary for global membrane support, since it is highly acrosome reaction, suggesting a possible role in an- polarized and lacking in large areas of plasma membrane of eucharyotic cells previously studied (Pollard, choring membrane protein domains. From the data presented on the distribution of actin 1984). The acrosomal cup of human spermatozoa was decoduring spermiogenesis and in ejaculated spermatozoa, it appears that actin serves a universal function during rated with antibodies against an immunoanalogue of , et al., 19841, the shaping of the acrosomal vesicle in mammalian erythrocyte a-spectrin ( ~ 2 3 0 Virtanen spermiogenesis. Despite this evidence, it seems that which indicated the major calmodulin binding polypepthe maintenance of this protein in particular regions of tide of these spermatozoa. A specific fluorescence was mature sperm heads or its loss is still an unanswered localized to the acrosome of the sea urchins and mice sperm, using an anti-fodrin antibody. Schatten et al. question. The aggregational state of actin represents another (1986)suggested that this protein, interacting with the debated point. Controversial observations are reported, plasma membrane and cortical actin, might stabilize such as S-1 decoration or microfilaments in rat and cytoskeletal-membrane components during fertilizaground squirrel spermiogenesis (Russel et al., 1986; tion. Moreover, spectrin linked with calmodulin and
Fig. 16. Immunostaining for spectrin-like proteins on testis Lowicryl sections. Anti-chicken erythrocyte spectrin antibody has been used and recognized with goat anti rabbit 5 nm. A, acrosome; N, nucleus. A Gold particles are present along the shaping acrosomal membranes (arrows) of this round spermatid. Bar = 0.17 km. B:
Spectrin is recognized by gold particles all along the shaping head membranes (arrows) of this elongated spermatid. Bar = 0.20 pm. C: Apparently the gold particles distribution (arrows) follows the profile of the acrosome in this spermatid. Bar = 0.12 pm.
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Fig. 17. Rabbit sperm cryosections immunostained for spectrin. Goat anti-rabbit 5 nm has been used as second antibody. IAM, inner acrosomal membrane; OAM, outer acrosomal membrane; PM,plasma membrane. A: Longitudinal section of a sperm head stained with anti-chicken erythrocyte spectrin. Gold particles are placed along the
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head membranes. Bar = 0.22 pm.B: In this cross-section through the four subacrosomal bulges (arrows), stained with anti-a-brain fodrin, gold particles follow the cytoplasmic faces of all the membranes. Bar = 0.20 pm.
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the cortical cytoskeleton plays a role in the spreading of mouse blastomeres (Sobel et al., 1988). The occurrence of a protein belonging to the “spectrin family” in mammalian spermatozoa was hypothesized for the properties of being a major calmodulin binding protein and of being a membrane-skeletal component of many eucharyotic cells. We have analyzed the presence of a spectrin-like protein in rabbit spermiogenesis and spermatozoa (Camatini and Casale, 1988) by SDS electrophoretic separation of sperm proteins and coelectrophoresis of chicken erythrocyte spectrin and sperm extract (Fig. 13) and by the use of anti-spectrin antibodies of different origin (anti-chicken spectrin, anti-brain fodrin, and anti-calf lens p230 spectrin). In immunoblots of sperm extract, the antibodies have recognized a band at the level of the 240 kDa protein subunit (Fig. 14A-C). A bright fluorescence at the acrosomal cap, the postacrosomal region, and the principal piece of the tail (Fig. 15A,B) is evident with the immunofluorescent technique. Moreover immunoelectron microscopy performed both on testis Lowicryl sections (Fig. 16A-C) and sperm cryosections (Fig. 17A,B) show that a spectrin-like protein is recognized by the antibodies used along the head membranes. Anti-spectrin label is on the cytoplasmic face of the PM, the OAM, and along the nuclear face of the IAM. These results suggest that a spectrin-like protein is a component of these membranes, from early (Fig. 16A,C) to mature spermatids (Fig. 16B).
CONCLUDING REMARKS A generalized cytoskeletal model for mammalian spermatozoa does not exist. We should think, rather than of a unique schematic drawing for ejaculated mammalian spermatozoa, of several combinations of different models in different species. It is reasonable to believe that the sperm in its mature form is modulated and maintained by cytoskeletal elements placed in correspondence to membranes, whose shape and function follow a species-specific model. Moreover, capacitation and fertilization events employ molecular mechanisms, which must be differently regulated in sperm belonging to the various species. For example, the migration of guinea pig and rabbit sperm antigens during capacitation is well documented, and epididymal guinea pig sperm do not contain actin (Halenda et al., 1987),while rabbit sperm do (Welch and O’Rand, 1985; Camatini et al., 1987). Several key questions are still unanswered. It is unknown whether the cytoskeletal proteins identified in spermatozoa, identical to these present in other cell systems, play the same functional role as in other cells or whether still unidentified elements are present. It is also unknown if different domains of the spermatozoa possess distinct cytoskeletal assemblies. Another puzzle emerges by the colocalization of some cytoskeletal elements and their regulatory proteins. For example, spectrin-actin, spectrin-calmodulin, and calmodulin-actin associations have been described both in gametes and in early developmental stages of embryos (Virtanen et al., 1984; Reima and Lehtonen, 1985; Camatini and Casale, 1987; Sobel et al., 1988).
Nonetheless, the relationships among such proteins are still unclear in extensively studied cell systems (Pollard, 1984). Evidence against the importance of spectrin-actin interaction is derived by the observation that no change in cell shape or actin distribution appears, when aggregation of spectrin is induced with anti-spectrin antibodies (Mangeat and Burridge, 1984). Moreover, the identification of a-spectrin as the common calmodulin binding subunit of erythroid and nonerythroid spectrin is still unclear (Lazarides and Nelson, 1982). Future research will clarify the molecular regulation of sperm compartmentalization if the efforts that already have obtained numerous encouraging results are focused on the study of the same model in its different aspects from the early stage until the fertilization process. It is perhaps a good suggestion to approach the cytoskeleton of spermatozoa as a system completely different from the well-characterized cytoskeleton of the other cells.
ACKNOWLEDGMENTS The authors are grateful to Dr. Giovanni Bernardini for his valuable suggestions during the preparation of the manuscript. Supported by M.P.I., 60%, 1989, C.N.R. Technology No. 9001981CTll. REFERENCES Bennet, V. (1985)The membrane skeleton of human erythrocytes and its implications for more complex cells. Annu. Biochem., 54:273304. Bloch, R.J., and Morrow, J.S. (1989)An usual p-spectrin associated with clustered acetylcholine receptors. J. Cell Biol., 108:481-493. Camatini, M., Anelli, G., and Casale, A. (1986a)Immunocytochemical localization of calmodulin in intact and acrosome-reacted boar sperm. Eur. J . Cell Biol., 41:89-96. Camatini, M., Anelli, G., and Casale, A. (1986b)Identification of actin in boar spermatids and spermatozoa by immunoelectron microscopy. Eur. J . Cell Biol., 42:311-318. Camatini, M.,and Casale, A. (1987)Actin and calmodulin coexist in the equatorial segment of ejaculated boar sperm. Gamete Res., 17: 97-105. Camatini, M., and Casale, A. (1988)Characterization of cytoskeletal elements in mammalian spermatozoa. In: Sacomeric and Non-Sarcomeric Muscle. U. Carraro, ed. Unipress, Padova, pp. 721-726. Camatini, M., Casale, A., Anelli, G., and Cifarelli, M. (1986~) Immunoelectron microscopic localization of actin in ionophore-treated boar sperm. Cell Biol. Int. Rep., 10231-238. Camatini, M., Casale, A., and Cifarelli, M. (1987)Immunoctyochemical identification of actin in rabbit spermiogenesis and spermatozoa. Eur. J. Cell Biol., 45:274-281. Camatini, M., Colombo, A., Bonfanti, P. (1991)Identification of spectrin and calmodulin in rabbit spermiogenesis and spermatozoa. Mol. Reprod. Dev. 28:62-69. Camatini, M., Franchi, E., and De Curtis, I. (1981)Permeability to lanthanum of blood testis barrier. Anat. Rec., 200:211-224. Casale, A,, Camatini, M., Skalli, O., and Gabbiani, G. (1988)Characterization of actin isoforms in ejaculated boar spermatozoa. Gamete Res., 20133-144. Clarke, G.N., Clarke, F.M., and Wilson, S. (1982)Actin in human spermatozoa. Biol. Reprod., 26:319-327. Clarke, G.N., and Yanagimachi, R. (1978)Actin in mammalian sperm heads. J. Exp. Zool., 205:125-132. Clermont, Y., and Leblond, C.P. (1955)Spermiogenesis in man, monkey, ram and other mammals as shown by periodic acid-Schiff techniques. Am. J . Anat., 96229-253. Damjanov, I., Damjanov, A., Lehto, V.P., and Virtanen, I. (1986)Spectrin in mouse gametogenesis and embryogenesis. Dev. Biol., 114: 132-140. Dym, M., andFawcett, D.W. (1970)The blood-testis in the rat and the
SPERMATOZOA CYTOSKELETAL ELEMENTS physiological compartmentation of the seminiferous epithelium. Biol. Reprod., 3:308-326. Fawcett, D.W. (1975) Ultrastructure and function of the Sertoli cell. In: Handbook of Physiology, Vol. 5. R.O. Greep, ed. Williams and Wilkins, Baltimore, pp. 21-55. Feinberg, J., Weinmann, J., Weinmann, S., Walsh, M.P., Harricane, M.C., Gabrion, J . , and Demaille, J.G. (1981) Immunocytochemical and biochemical evidence for the presence of calmodulin in bull sperm flagellum. Biochim. Biophys. Acta, 673:303-311. Flaherty, S.P., Breed, W.G., and Sarafis, V. (1983) Localization of actin in the sperm head of the plains mouse. J. Exp. Zool., 225: 497-500. Flaherty, S.P., Winfrey, V.P., and Olson, G.E. (1986) Localization of actin in mammalian spermatozoa: A comparison of eight species. Anat. Rec., 216:504-515. Flaherty, S.P., Winfrey, V.P., and Olson. G.E. (1988) Localization of actin-in human, bull, rabbit, and hamster sperm by immunoelectron microscopy. Anat. Rec., 221:599-610. Fouquet, J.P., Kann, M.-L., and Dadoune, J.-P. (1989) Immunogold distribution of actin during spermiogenesis in the rat, hamster, monkey, and human. Anat. Rec., 223:35-42. Gaunt, S.J., Brown, C.R., and Jones, R. (1983) Identification of mobile and fixed antigens on the plasma membrane of rat spermatozoa using monoclonal antibodies. Exp. Cell Res., 144:275-284. Glenney, J.R., Jr., and Glenney, P. (1983) Fodrin is the general spectrin-like protein found in most cells whereas spectrin and the TW protein have a restricted distribution. Cell, 34503-512. Gundersen, G.G., Kalnoski, M.H., and Bulinsky, J.C. (1984) Distinct populations of microtubules: Tyrosinated and nontyrosinated alpha tubulin are distributed differently in vivo. Cell, 38:779-789. Halenda, R.M., Primakoff, P., and Myles, D.G. (1984) Evidence for the presence of actin in guinea pig spermatogenic cells and its loss prior to formation of mature sperm. J. Cell Biol., 99:394a. Halenda, R.M., Primakoff, P., and Myles, D.G. (1987) Actin filaments, localized to the region of the developing acrosome during early stages, are lost during later stages of guinea pig spermiogenesis. Biol. Reprod., 36:491-499. Hopper, J.E., and Kelly, R.B. (1984) Calmodulin is tightly associated with synaptic vesicles independent of calcium. J . Biol. Chem., 259: 148-153. Jones, H.P., Bradford, M.M., McRorie, R.A., and Cormier, M.J. (1978) High levels of calcium-dependent modulator protein in spermatozoa and its similarity to brain modulator protein. Biochem. Biophys. Res. Commun., 82:1264-1272. Jones, H.P., Lenz, R.W., Palevitz, B.A., Cormier, M.J. (1980) Calmodulin localization in mammalian spermatozoa. Proc. Natl. Acad. Sci. U.S.A., 77:2772-2776. Korn, E.D. (1982) Actin polymerizaiton and its regulation by proteins from nonmuscle cells. Physiol. Rev., 62:672-737. Lakoski, K., Williams, C., and Saling, P. (1989) Proteins of the acrosomal region in mouse sperm: Immunological probes reveal posttesticular modifications. Gamete Res., 23:21-37. Lazarides, E., and Nelson, W.J. (1982) Expression of spectrin in nonerythroid cells. Cell, 31505-508. Longo, F.J., Krone, G., and Franke, W.W. (1987) Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: A novel class of cytoskeletal elements. J . Cell Biol., 105:1105-1120. Mangeat, P.H., and Burridge, K. (1984) Immunoprecipitation of nonerythrocyte spectrin within live cells following microinjection of spectrin antibodies: Relation to cytoskeletal structures. J . Cell Biol., 981363-1377. Marchesi, V.T. (1985) Stabilizing infrastructure of cell membranes. Annu. Rev. Cell Biol., 1:531-562. Masri, B.A., Russel, L.D., and Vogl, A.W. (1987) Distribution of actin in spermatids and adjacent Sertoli cell regions of the rat. Anat. Rec., 218:20-26. Mercier, F., Reggio, H., Devilliers, G., Bataille, D., and Mangeat, P. (1989) Membrane-cytoskeleton dynamics in rat parietal cells: Mobilization of actin and spectrin upon stimulation of gastric acid secretion. J. Cell Biol., 108:441-453. Miller, L.M., Kalnoski, M., Yunossi, Z., Bulinski, J.C., and Reisler, E. (1987) Antibodies derected against N-terminal residues on actin do not block acto-myosin binding. Biochemistry, 26:6064-6070. Morrow, M.S. (1989) The spectrin membrane skeleton: Emerging concepts. Curr. Opin. Cell Biol., 1:23-29. Myles, D.G., F’rimakoff, P., and Bellv6, A.R. (1981) Surface domains of the guinea pig sperm defined with monoclonal antibodies. Cell, 23: 433-439.
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Noland, T.D., Van Eldik, L.J., Garbers, D.L., and Burgess, W.H. (1985) Distribution of calmodulin and calmodulin-binding proteins in membranes from bovine epididymal spermatozoa. Gamete Res., 11:297-303. Ochs, D., and Wolf, D.P. (1985) Actin in ejaculated human sperm cell. Biol. Reprod., 33:1223-1226. Ochs, D., Wolf, D.P., and Ochs, R.L. (1986) Intermediate filament proteins in human sperm heads. Exp. Cell Res., 167:495-504. Olson, G.E., Winfrey, V.P., and Flaherty, S.P. (1987) Cytoskeletal assemblies of mammalian spermatozoa. Ann. N.Y. Acad. Sci., 29: 222-246. Olson, G.E., Winfrey, V.P., Garbers, D.L., and Noland, T.D. (1985) Isolation and characterization of a macromolecolar complex associated with the outer acrosomal membrane of bovine spermatozoa. Biol. Reprod., 33:761-779. O’Rand, M., and Fisher, S. (1987) Localization of zona pellucida binding sites on rabbit spermatozoa and induction of the acrosome reaction by solubilized zonae. Dev. Biol., 119551-559. O’Rand, M.G., Matthews, J.E., Weech, J.E., and Fisher, S.J. (1985) Identification of mna binding proteins of rabbit, pig, human, and mouse spermatozoa on nitrocellulose blots. J. Exp. Zool., 235423428. Otey, C.A., Kalnosky, M.H., and Bulinski, J.C. (1986)A procedure for the immunoblotting of proteins separated on isoelectric focusing gels. Anal. Biochem., 157:71-76. Pelletier, R.M. (1988) Cyclic modulation of Sertoli cell junctional complexes in a seasonal breeder: The mink (Mustela vision). Am. J . Anat., 183:68-102. Peterson, R.N., Bozzola, J.J., Hunt, W.P., and Darabi, A. (1990) Characterization of membrane-associated actin in boar spermatozoa. J. Exp. Zool., 253:202-204. Peterson, R.N., Chaundhry, P., and Tibbs, B. (1989) Calcium-binding protein of boar spermatozoan plasma membranes: Identification and partial characterization. Gamete Res., 23:49-60. Phillips, M.D. (1972) Substructure of the mammalian acrosome. J. Ultrastruct. Res., 38:591-604. Pollard, T.D. (1984) Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins. J. Cell Biol., 99:19701980. Pollard, T.D., and Cooper, J.A. (1986) Actin and actin-binding proteins. A critical evolution of mechanism and functions. Ann. Rev. Biochem., 55:987-1035. Primakoff, P., and Myles, D.G. (1983) A map of the guinea pig sperm surface constructed with monoclonal antibodies. Dev. Biol., 98:417428. Reima, I., and Lehtonen, E. (1985) Localization of nonerytroid spectrin and actin in mouse oocytes and preimplantation embryos. Differentiation, 30:68-75. Russel. L.D.. Goh. J.C. Rashed. R.M.A.. and Vod. A.W. (1988) The consequences of actin disruption a t Sertoli ec6plasmic specialization sites facing spermatids after in vivo exposure of rat testis to cytochalasin D. Biol. Reprod., 39:105-118. Russel, L.D., and Peterson, R.N. (1985) Sertoli cell junction: Morphological and functional correlates. Int. J . Cyt., 94:177-211. Russel, L.D., Weber, J.E., and Vogl, A.W. (1986) Characterization of filaments within the subacrosomal space of rat spermatids during spermiogenesis. Tissue Cell, 183387-898. Schatten, H., Cheney, R., Balczon, R., Cline, C., Simerly, C., and Schatten, G. (1986) Localization of fodrin during fertilization and early development of sea urchin and mice. Dev. Biol., 118:457-466. Sobel, J.S., Goldstein, E.G., Venuti, J.M., and Welsh, J.M. (1988) Spectrin and calmodulin in spreading mouse blastomeres. Dev. Biol., 126:47-56. Talbot, P., and Kleve, M.G. (1978) Hamster sperm cross react with anti-actin. J. Exp. Zool., 204:131-136. Tamblyn, T.M. (1980) Identification of actin in boar epididymal spermatozoa. Biochem. Biophys. Res. Commun., 62:328-335. Van Eldik, L.J., and Burgess, W.H. (1983) Analytical subcellular distribution of calmodulin and calmodulin-binding proteins in normal and virus-transformed fibroblasts. J. Biol. Chem., 258:4539-4547. Virtanen, I., Badley, R.A., Paasivuo, R., and Lehto, V.P. (1984) Distinct cytoskeletal domains revealed in sperm cells. J. Cell Biol., 99:1083-1091. Vogl, A.W. (1988) Changes in the distribution of microtubules in rat Sertoli cells during spermagogenesis. Anat. Rec., 222:34-41. Vogl, A.W. (1989) Distribution and function of organized concentra-
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tions of actin filaments in mammalian spermatogenic cells and Sertoli cells. Int. Rev. Cyt., 119:l-56. Vogl, A.W., Grove, B.D., and Lew, G.J.(1986) Distribution of actin in Sertoli cell ectoplasmic specializations and associated spermatids in the ground squirrel testis. Anat. Rec., 215331-341. Weinman, S., Ores-Carton, C., Escaig, F., Feinberg, J., and Puszkin,
S. (1986) Calmodulin immunoelectron microscopy: Redistribution during ram spermatogenesis and epididymal maturation. II. J. Histochem. Cytochem., 32:1181-1193. Welch, J.A., and O’Rand, M.G. (1985) Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit. Dev. Biol., 109:411-417.
Cytoskeletal Elements in Mammalian Spermiogenesis and Spermatozoa MARINA CAMATINI, ANITA COLOMBO, AND PATRIZZA BONFANTI Department of Biology, University of Milano, I-20133 Milan, Italy
KEY WORDS
Spermiogenesis, Spermatozoa, Mammals, Cytoskeleton
ABSTRACT Identification of the cytoskeletal elements and their role in the formation as well as the maintenance of head membrane compartmentalization is a much debated issue in mammalian spermatozoa. Data which have emerged during the last ten years are summarized. Those which have converged in a common opinion, such as the distribution of actin in mammalian spermiogenesis, are distinguished from those which have to be confirmed, such as the role of actin related proteins and actin in mature spermatozoa. SPERM MORPHOGENESIS The transformation of the spermatid into the spermatozoon is a complex process that proceeds through a series of cytological changes designated as “steps)’(Clermont and Leblond, 1955). These steps can be grouped into phases whose names are descriptive of the principal activities that characterize them. The first steps constitute the Golgi phase during which the Golgi apparatus begins the production of the acrosomal vesicle a t the side of the nucleus facing the lumen of the seminiferous tubule. This marks the anterior end of the future head of the sperm. At the opposite side of the spermatid, the centrioles begin to organize the tail fibers. During the cup phase, the acrosome spreads over the surface of the condensing nucleus; the spermatid rotates, so that the acrosomal complex faces the basement membrane of the seminiferous tubule and the developing tail faces the lumen. During the acrosome phase, the nucleus elongates and the cytoplasm is displaced, posteriorly, towards the growing flagellum, leaving, anteriorly, the acrosome covered only by the plasma membrane. An equatorial ring of fibrous material around the nucleus serves as the origin of an array of microtubules, the manchette, which extends as a cylinder into the distal cytoplasm. The manchette is involved in the displacement of the cytoplasm of the spermatid, and it disappears when the cytoplasm recedes from the nucleus. The final steps of spermatogenesis constitute the maturation phase. The nucleus becomes flattened and highly condensed, the centriole and columns of the connecting piece appear, and the mitochondria become organized around the proximal part of the flagellum to form the midpiece. Cytoskeletal Elements Involved in Sperm Morphogenesis It is a t present well known that cytoskeletal elements such as actin filaments (Masri et al., 1987; Vogl, 19891, microtubules (Gundersen et al., 1984; Vogl, 1988), and regulating proteins such as spectrin (Glenney and Glenney, 1983) and caImodulin (Camatini et al., 1986a; Weinman et al., 1986) are involved in cyto-
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plasm redistribution inside the cell, in cell shaping, and in morphogenetic movements of differentiating cells. The analysis of the events which regulate such phenomena may be performed by exploring the localization of cytoskeletal elements involved in cell shaping by the immunoelectron-microscopic technique. This technique employs antibodies against cytoskeletal proteins (Otey et al., 1986; Miller et al., 19871, which are characterized by immunoblots of one-dimensional (1D) or two-dimensional (2D) gel electrophoresis of the proteins under analysis. These antibodies are then used on fixed cells and processed for immunofluorescence, or on Lowicryl sections and cryosections and processed for immunoelectron microscopy. The antibodies can be recognized by specific anti-IgG conjugated with fluorescein or with colloidal gold particles (Carnatini et al., 1986a,c). Spermatogenesis is characterized by spectacular shape changes of both the nucleus and the cytoplasm. These changes are responsible for the final polarized form achieved in mature spermatozoa. Shape transformations are mediated by cytoskeletal elements, present inside the cell, which provide cytoplasmic tranlocation and organelle redistribution. This is the reason actin organization and the manchette of microtubules have been extensively analyzed in spermatogenic cells. While the role of microtubules is actually well defined, the presence of other cytoskeletal elements inside the spermatogenic cells has been long and controversially analyzed (Russell et al., 1986; Halenda et al., 1984, 1987). Numerous reports agree on the involvement of actin in sperm head morphogenesis (Vogl, 1989). Actin is certainly present in the spermatogenic cells themselves, and in attached Sertoli cell ectoplasmic specializations of all the mammalian species examined (see Camatini et al., 1987; Vogl, 1989; Fouquet et al., 1989).
Received April 24, 1990 accepted in revised form June 26,1990. Address reprint requests to Prof. Marina Camatini, Dipartmento di Biologia, Via Celoria 26, 1-20133 Milano, Italia.
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Fig. 1. Electrophoretic analysis (a) and immunoblot (b) of calmodulin (A) performed also in the presence of 1 mM Caf (B)or 5 m M EGTA (C). A a: PAGE profile (15%) of calmodulin from bovine testis. b Markers (lane l), polypeptides of boar sperm (lane Z), and purified calmodulin (lane 3) run in PAGE (15%), blotted, and stained with rabbit antiserum (lanes 4,5) and with anti-sheep antibodies (lanes 6, +
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7). B-C: SDS-PAGE (15% wiv) of markers (lane l), sperm extract (lane 21, and bovine testis calmodulin (lane 3) stained with 1% comassie brilliant blue; sperm extract (lane 4) and bovine testis calmodulin (lane 5) run in SDS-PAGE 15%, blotted onto nitrocellulose paper, and immunostained for calmodulin.
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Among these events, the movement of spermatocytes through the blood-testis barrier (Dym and Fawcett, 1970; Fawcett, 1975; Camatini et al., 1981; Pelletier, 1988), the translocation of spermatids to the luminal surface of the tubule, and the release of spermatozoa from the epithelium are the most significative ones. Although the underlying mechanism of these events is still unknown, the complex cytoskeletal network of Sertoli cells as well as the inter-Sertoli junctions are likely to be involved. Microfilaments (Russell et al., 1988; Vogl, 1989) and microtubules (Russell and Peterson, 1985) of Sertoli supporting cells during the development of spermatids and spermatozoa have been extensively studied, and their role is now well defined. Thus, sperm morphogenesis appears to be mediated by cytoskeletal elements in the supporting Sertoli cells as well as by cytoskeletal elements in specific regions of the shaping sperm heads.
Fig. 2. Fluorescent localization of calmodulin in boar (A-C) and rabbit (D-E) spermatozoa. A-B: Paraformaldehyde-fixed sperm. Fluorescence is on the acrosomal cap, the basal and middle piece of the tail. Bar = 5 pm. C: Methanol-fixed sperm. Fluorescence is present on the equatorial segment (arrowhead). Bar = 5 pm. D-E The subacrosomal bulges, the anterior part of the postacrosomal region, and the basal and middle piece of the tail are brightly fluorescent. F Phasecontrast image of E. Bar = 10 pm.
Role of Sertoli Cells in Sperm Morphogenesis Sertoli cells are thought t o be involved with many of the morphogenetic events that occur in the mammalian seminiferous epithelium during spermiogenesis.
ACTIN AND REGULATORY PROTEINS Among the cytoskeletal proteins, actin is the one that has received major attention during the past ten years (Pollard and Cooper, 1986). Owing to its importance in biology, it has been studied in considerable detail. First, it seemed that the discovery of the major class of actin-binding proteins could elucidate the assembly and function of the actin system in cells. Second, it was apparent that more complicated mechanisms must be involved. Although most of the regulatory proteins have been localized in cells, there is almost no direct experimental evidence regarding the function of such regulatory proteins in living cells. There are a t least four different families of proteins that bind primarily to actin monomers (Pollard and Cooper, 1986) and every one of the actin monomer binding proteins studied to date forms a complex with actin that does not polymerize, as free actin monomers do. This inhibition is explained by the ability of these proteins to sequester monomers in a nonpolymerizable complex. Comparison of the primary structure of actin shows that this protein has been highly conserved during evolution (Korn, 1982). There are less than 5% differences in the amino acid sequence of muscle and cytoplasmic actins from protozoa to vertebrates; plants have multiple actins that differ from each other and from animal actins by more than 10%in the amino acid sequence. Based on the amino acid sequence homologies, all Of invertebrates appear to be direct descendants of the cytoplasmic actins found in protozoa and fungi, while the muscle actins of vertebrates form an easily distinguishable family. Despite these differences in actin isoformsexpressed, it has long been clear that actin plays a central role in motility beyond its classic contractile function in muscle. Membrane-Skeleton in Non-Muscle Cells The role of actin in non-muscle cells remains an intriguing puzzle. Recent findings and new approaches (Bennett, 1985; Marchesi, 1985; Mercier et al., 1989) have elucidated the composition of a membrane-bound
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Fig. 3. Sections of boar (A-C)and rabbit (D) sperm heads immunostained for calmodulin, detected with antibody conjugated to gold particles. These are distributed inside the acrosome (A), along the equatorial segment (E)and the postacrosomal region (PA). Arrowheads evidence the stain along the E segment (B). A: Bar = 0.45 pm.
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B: Bar = 0.15 pm. The head of a late spermatid shows gold particles inside the acrosome and along the microfilaments (arrows) of the surrounding Sertoli cell (C). In this longitudinal section (D) the particles are present on the postacrosomal region (PA) and the subacrosomal bulges. E, equatorial segment; N, nucleus. C-D: Bar = 0.29 pm.
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Fig. 4. Specificity of anti-actin antibodies on different actins and boar sperm extracts. Amido black staining (A) and immunostaining (€3-C) of boar spermatozoa (lanes 1) and purified muscle actin (from rabbit skeletal muscle) (lanes 2), purified smooth-muscle actin (from
chicken gizzard) (lanes 3), and purified non-muscle actin (from Pfiysarum) (lane 41, blotted to nitrocellulose. A Polypeptides run on 10% SDS-PAGE. B: Immunostaining with the anti-y antibody. C: Immunostaining with the anti-a antibody.
lattice. Among these studies, the cytoskeletal network, underlying the erythrocyte membrane a t the molecular level, has received considerable attention, and the model of the membrane-skeleton, which gives the erythrocyte its stability and unique viscoelastic properties, has been well defined (Bennett, 1985; Marchesi, 1985). In search of the supramolecular organization of the erythrocyte membrane-skeleton, extensive studies have been directed a t the structure and chemical nature of spectrin, including its binding with other proteins, such as actin, ankyrin, and band 4.1 (Morrow, 1989).The erythrocyte membranes have played a leading role as a model system in research on the attachment of the cytoskeleton to the membrane. However, the applicability of this system to other cell types remains a matter for speculation. The hypothetical model of the membrane-skeleton of the nicotinic acetylcholine receptor (Bloch and Morrow, 1989) involves oligomeric actin which cross-links spectrin. Thus, it is apparent that these two proteins are gaining attention when receptor organization, topographic membrane assembly, and membrane trafficking appear to be functions expressed in non-erythroid cells.
al., 1981; Primakoff and Myles, 1983; O’Rand et al., 1985; O’Rand and Fisher, 1987). It has been reported that plasma membrane proteins in the postacrosomal (PA) region exibit little lateral mobility in the rabbit mature spermatozoa in contrast to the lateral mobility of proteins in the plasma membrane of the acrosomal region (Welch and O’Rand, 1985). Identical observations have been made in guinea pig (Primakoff and Myles, 1983) and rats (Gaunt et al., 1983). These results imply an anchoring mechanism below the plasma membrane in particular head regions. If this assumption is correct, the colocalization in gametes of spectrin and calmodulin (Virtanen et al., 1984; Sobel et al., 1988; Camatini et al., 1991), of actin and calmodulin (Camatini and Casale, 19871, and of spectrin and actin (Reima and Lehtonen, 1985) suggests that they might have a cytoskeletal-membrane role. Besides these regulatory proteins, other cytoskeletal elements have been described in mammalian spermatozoa. Intermediate filament proteins, such as vimentin (Virtanen et al., 1984; Ochs et al., 1986; Olson et al., 1987) and keratin (Ochs et al., 1986), were found in human spermatozoa. Moreover, Longo et al. (1987) have described a novel class of cytoskeletal proteins which surround the nuclei of bull and rat spermatozoa.
Membrane-Cytoskeletal Elements in Sperm Heads The mammalian sperm head represents a fascinating system for the compartmentalization of its regions, performed by the plasma membrane (PM), the outer acrosomal membrane (OAM), and the inner acrosomal membrane (IAM). Protein mobility appears to be restricted in particular areas of these membranes, which are maintained after the acrosome reaction (Myles et
IDENTIFICATION OF CYTOSKELETAL ELEMENTS IN SPERMIOGENESIS AND SPERMATOZOA During the last ten years, numerous laboratories have directed their research at the analysis of cytoskeleta1 elements involved in the compartmentalization of
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Fig. 5. Two-dimensionalgels (A,B) and immunoblots(C,D) of rabbit sperm extracts. A: 2D electrophoresis of sperm extract. B: Comigration of sperm extract and chicken gizzard actin. The presumptive y-actin spot (white arrowhead) and p-actin spot (black arrowhead)
are circled anti-y (C) and anti-a (D)antibodies show one spot each. The cathode (-1, the anode (+), and pH range (6.5-4.5) are indicated. Arrow: 45,000 molecular weight.
the head membranes of mammalian spermatozoa with different immunocytochemical techniques. The distribution of several cytoskeletal proteins in mammalian spermiogenesis and spermatozoa have been described in numerous species (see Olson et al., 1987). Actin has received considerable attention, and its presence during spermiogenesis is well documented (see Camatini et al., 1986b, 1987; Vogl, 19891, since a consistent distribution pattern has emerged. Despite uniform results obtained with various techniques, it is reported that actin is not maintained in all mature spermatozoa of the species examined. Moreover, it appears that actin is not placed in the same cell compartments. Thus, a common functional role may be suggested for actin during spermiogenesis, while it remains a question for mature spermatozoa.
Among proteins directly or indirectly associated with actin, calmodulin and spectrin are the ones examined by several laboratories.
Calmodulin Calmodulin was first demonstrated in the soluble fraction of mammalian sperm (Jones et al., 1978) and recently it has been found associated with the PM proteins of bovine epididymal spermatozoa both in the presence of EGTA and C a i + (Noland et al., 1985; Peterson et al., 1989). Moreover, it has been suggested (Olson et al., 1985) that calmodulin may be bound to the OAM complex and is released during Ca+ influx. These biochemical findings demonstrate the presence of multiple classes of calmodulin binding proteins in mammalian sperm as well as in other cells (Van Eldik +
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Fig. 6. Localization of actin on early boar spermatids. Testis Lowicryl sections are stained with an anti-actin monoclonal antibody, recognized with goat anti-mouse 10 nm gold particles. Actin (arrowheads) is visible on the subacrosomal area (A-D) and on the microfilaments (arrow) of Sertoli cell (S) specializations (B-D). A, acrosome; N, nucleus. A: Bar = 0.71 km. B: Bar = 0.29.C: Bar = 0.35 pm. D: Bar = 0.40 pm.
Fig. 7. Localization of actin on early rabbit spermatids. Testis Lowicryl sections are stained with the anti-cx actin antibody, evidenced with goat anti-rabbit 10 nm (A-C) and the monoclonal antiactin antibody, evidenced with goat anti-mouse 5 nm (D.) Actin distribution is visible in the subacrosomal area (A-C, arrowheads). The particles are continuous in the thin space between the acrosome and
the nucleus (AX). When the spermatid head flattens (D), particle density in the subacrosomal area is lower. A, acrosome; N, nucleus. Arrows indicate the final region of contact between the acrosomal vesicle and the nucleus. A: Bar = 0.9 km. B: Bar = 0.4 Fm. C: Bar = 0.38 pm. D: Bar = 0.26 pm.
Fig. 8. Localization of actin on boar late spermatids (arrowheads) and on Sertoli cell specializations (arrows). N, nucleus. In longitudinal sections (A) spermatid heads present discontinuous particles under the IAM (arrowheads). The label, which caps the heads, corresponds to Sertoli (S) microfilaments. A Sertoli-Sertoli junctional specialization (double arrow) is indicated. A Bar = 0.90 pm. Spermatid cross-sections a t the acrosome level (B,C,E) and a t the PA re-
gion (D). B Bar = 0.24 pm. C:Bar = 0.26 pm. D: Bar = 0.50 pm. E: Bar = 0.23 km. The particles are present under the plasma membrane in the postacrosomal region of a mature spermatid (D). In a longitudinal tangential section (F),the mature spermatid is enwrapped by Sertoli specializations, marked with gold particles. F: Bar = 0.33 pm.
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Fig. 9. Localization of actin in rabbit late spermatids (A-E).Actin immunostaining is performed with the monoclonal anti-actin antibody recognized by goat anti-mouse 5 nm (C,D) or goat anti-mouse 10 nm (A) and anti-ol antibody is recognized by goat anti-rabbit 10 nm (B,D). A, acrosome; N, nucleus; PA, postacrosomal region. A Bar = 0.25 pm. B: Bar = 0.3 km. C: Bar = 0.18 km. Arrowheads indicate
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the staining on the subacrosomal and postacrosomal region; arrows indicate the Sertoli cell (S)specialization. Discontinuous particles are present in the subacrosomal area (A,B,D). The postacrosomal region is marked by particles close to the plasma membrane (0. The label on surrounding microfilaments parallels the membrane of the spermatids (D,E). D: Bar = 0.29 pm. E: Bar = 0.45 *m.
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and rabbit (Fig. 2D,E) spermatozoa. Immunoelectron microscopy furnished additional details (Weinman et al., 1986; Camatini et al., 1986a) and calmodulin appears to be localized along the postacrosomal membrane (PA) and the cytoplasmic face of the OAM of the equatorial (E) segment in boar (Fig. 3A,B) and rabbit (Fig. 3D) spermatozoa. Moreover, calmodulin had been localized in boar (Fig. 3C) and bull (Weinman et al., 1986) spermatids. Rabbit spermatozoa, which have four subacrosomal bulges (Phillips, 1972), have calmodulin also in these regions (Fig. 3D). The distribution of calmodulin in mammalian spermatozoa supports the idea that this protein is related to the acrosome reaction, playing a key role in the fertilization process.
Fig. 10. Immunofluorescence localization of actin on boar (A-B) and rabbit (D) spermatozoa. Fluorescence is present at the curved region of the heads, at the equatorial segment, and at the pericentriolar area (A). Sperm, which have lost their acrosomes,present a weak fluorescent line at the IAM (arrows) (B). The absence of label on sperm stained with rhodamine-phalloidin is evident (C). Fluorescent (D) and corresponding phase-contrast(E) images of rabbit sperm. The postacrosomal regions (D) and the subacrosomal bulges are brightly fluorescent (arrow). A-E: Bar = 10 pm.
and Burgess, 1983; Hopper and Kelly, 1984). The specificity of the anti-calmodulin antibodies we used was tested by immunostaining blots of sperm extracts and purified calmodulin (Fig. 1A); the presence of a single band in the 17 kDa region confirms the reactivity of the antibody. Thus, biochemical results demonstrate the existence of calmodulin and its different electrophoretic mobility in the presence of Ca ' (Fig. 1B) or EGTA (Fig. 1 0 . An indirect immunofluorescence technique showed calmodulin in ejaculated guinea pig (Jones et al., 1980), bull (Feinberg et al., 19811, boar (Figs. 2A-0, +
Actin Actin in ejaculated mammalian spermatozoa has been studied by numerous authors (Talbot and Kleve, 1978; Clarke and Yanagimachi, 1978; Tamblyn, 1980; Clarke et al., 1982; Flaherty et al., 1983, 1986, 1988; Virtanen et al., 1984; Welch and ORand, 1985; Ochs and Wolf, 1985; Camatini et al., 1986b,c; 1987; Camatini and Casale, 1987; Casale et al., 1988; Peterson et al., 1990). The difficulties encountered in its localization, using antibodies of different specificities with immunofluorescence, with immunoelectron microscopy, and with biochemical methods, are certainly related to the small amount of actin in these cells. Comparing the results obtained with anti-actin antibodies directed against actins of various origin (Fig. 4A-C), we have demonstrated that the differences in antibody reactivity depend on the relative proportion of the a,(3, and y isoforms present in the actins examined. Actin was extracted from human spermatozoa (Ochs and Wolf, 19851, extracted and identified on 2D PAGE immunoblots as a single spot of PI = 5.45 in rabbit spermatozoa (Welch and ORand, 1985). We have obtained 2D gel separation of actin in boar (Casale et al., 1988) and rabbit spermatozoa (Fig. 5A,B), and we have identified in immunoblots of these gels two spots (Fig. 5C,D) corresponding to y and p isoforms of actin. Thus, we can assume that rabbit and boar spermatozoa express the P and y isoforms usually present in nonmuscle cells. The morphological results have shown that actin is present from the early cup phase of spermiogenesis in ground squirrel (Vogl et al., 19861, rat (Russell et al., 1986; Vogl et al., 19861, guinea pig (Halenda et al., 19871, boar (Fig. 6A-D) and rabbit (Fig. 7A-D). The shaping of the acrosome around the anterior pole of the nucleus apparently follows an identical scheme in all mammals studied so far. Actin becomes apparent when the acrosomal vesicle makes contact with the anterior pole of the nucleus. As has been shown with different immunostaining techniques, the restricted area of juxtaposition between the flattening membrane and the nuclear envelope is always positive to the stain. Immunoelectron microscopy shows that the restricted area of juxtaposition between the flattening membrane and the nuclear envelope is labelled by anti-actin antibody. This label parallels the shaping of the vesicle over the nucleus (Figs. 6, 7, A,B). Apparently, the inner acroso-
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Fig. 11. Ejaculated boar sperm stained with a monoclonal antiactin antibody evidencedwith goat anti-mouse 10 nm. A, acrosome;E, equatorial segment; PA, postacrosomal region. The longitudinal tangential section of the sperm head (A) shows gold particles on the E
segment. A: Bar = 0.71 pm. Particles are aligned between the OAM of the equatorial segment and the PM, in a cross-sectionedhead. B: Bar = 0.29 km. The sagittally sectioned head (C) is labeled only at the equatorial segment. C: Bar = 0.38 km.
ma1 membrane and the nuclear envelope are connected by actin for the entire duration of their contact. Then chromatin condenses and the spermatid head flattens (Fig. 8A-F). The thin space, separating the acrosomal vesicle from the nucleus, maintains the same thickness until the spermatids mature, even though it is less marked by gold particles (Figs. 8B-E, 9B,D). This observation might account for a changed distribution of actin in late spermatids, or for a complete loss of it (Halenda et al., 1987). Actin filaments in Sertoli cell specializations become more evident in coincidence with spermatid elongation, since ectoplasmic junctional areas expand to surround all the spermatid head (Figs. 8C-F, 9B,D,E). Actin is maintained in correspondence of the equatorial segment and the postacrosomal region after ejaculation in human (Clarke et al., 1982; Virtanen et al.,
1984; Ochs and Wolf, 19851, boar (Tamblyn, 1980; Camatini et al., 1987; Peterson et al., 1990), bull, hamster (Flaherty et al., 1988), and rabbit (Welch and ORand, 1985; Camatini et al., 1987) spermatozoa. However, its conservation in ejaculated spermatozoa does not seem to be the rule. In some cases it is lost during maturation, as reported for rat late spermatids (Russell et al., 19861, for plains mouse epididymal spermatozoa (Flaherty et al., 1986), and for guinea pig mature sperm (Halenda et al., 1987). Immunofluorescence has been the technique most used in actin detection, even though it cannot account for a precise localization (Fig. 10A-D). The immunoelectron microscopic technique has furnished details on the distribution of this protein in boar (Fig. 11A-C) and rabbit (Fig. 12A-D) spermatozoa. Actin appears to be conserved in particular head membrane regions, which will be maintained after the
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Fig. 12. Actin localization on rabbit ejaculated sperm (A-D). Actin is detected with the anti-a antibody recognized with goat antirabbit 10 nm in A and it is detected with the monoclonal antibody recognized with goat anti-mouse 15 nm in B and C. Arrows, gold particles; E, equatorial segment; arrowheads, subacrosomal bulges;
PA, postacrosomal region. Longitudinal sections show particles on the subacrosomal bulges (A,B,D) and on the anterior part of the postacrosomal region. A: Bar = 0.31 km. B: Bar = 0.33 km. D: Bar = 0.15 wm. The anterior part of t h e postacrosomal region presents clusters of particles. C: Bar = 0.23 Fm.
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Fig. 15. Fluorescent localization of anti-chicken erythrocyte spectrin (A)and anti-a-mouse brain fodrin (B) antibodies. Fluorescence is on the acrosomal cap, the subacrosomal bulges, and the principal piece of the tail.
Vogl et al., 1986),rhodamine-phalloidin stain in rabbit spermiogenesis (Welch and O'Rand, 1985), and protofilaments associated with boar membrane spermatozoa (Peterson et al., 1990). In the species we examined (Camatini et al., 1986b; 1987), actin is present in oligomeric form and in correspondence with membrane regions, whose domains must be anchored. Fig. 13. SDS electrophoretic separation on 7.5% slab gel of high molecular weight markers (M),brain extract (lane l), sperm extract (lane 2), chicken erythrocyte spectrin (lane 3), and sperm extract run with purified spectrin (lane 4). Numbers indicate the protein marker molecular weight in kDa.
Spectrin Evidence has emerged recently on the presence of a spectrin-like protein in gametes, zygotes, and embryos of sea urchins and mice (Schatten et al., 1986; Lakoski Fig. 14. Blots of electrophoretically separated proteins from brain et al., 1989), in ejaculated human (Virtanen et al., (lane 1)and sperm (lane 2) extracts probed with different antibodies. 1984) and rabbit (Camatini and Casale, 1988; CamaA Anti-chicken spectrin. B: Anti-mouse brain a-fodrin. C:Anti-calf lens p230 spectrin. D Detection of calmodulin binding proteins on tini et al., 1991) spermatozoa, and in mouse gametogesperm polypeptides, after electrophoresis and transfer onto nitrocel- nesis (Damjanov et al., 1986).While the role of spectrin lulose paper. The blots were incubated in the presence of either 1mM and related proteins in forming the membrane-skelCaC12 (lane 1)or 5 mM EGTA (lane 2). eton in the erythrocytes is well defined, in non-erythroid cells it is less clear. Spectrin seems to be unnecessary for global membrane support, since it is highly acrosome reaction, suggesting a possible role in an- polarized and lacking in large areas of plasma membrane of eucharyotic cells previously studied (Pollard, choring membrane protein domains. From the data presented on the distribution of actin 1984). The acrosomal cup of human spermatozoa was decoduring spermiogenesis and in ejaculated spermatozoa, it appears that actin serves a universal function during rated with antibodies against an immunoanalogue of , et al., 19841, the shaping of the acrosomal vesicle in mammalian erythrocyte a-spectrin ( ~ 2 3 0 Virtanen spermiogenesis. Despite this evidence, it seems that which indicated the major calmodulin binding polypepthe maintenance of this protein in particular regions of tide of these spermatozoa. A specific fluorescence was mature sperm heads or its loss is still an unanswered localized to the acrosome of the sea urchins and mice sperm, using an anti-fodrin antibody. Schatten et al. question. The aggregational state of actin represents another (1986)suggested that this protein, interacting with the debated point. Controversial observations are reported, plasma membrane and cortical actin, might stabilize such as S-1 decoration or microfilaments in rat and cytoskeletal-membrane components during fertilizaground squirrel spermiogenesis (Russel et al., 1986; tion. Moreover, spectrin linked with calmodulin and
Fig. 16. Immunostaining for spectrin-like proteins on testis Lowicryl sections. Anti-chicken erythrocyte spectrin antibody has been used and recognized with goat anti rabbit 5 nm. A, acrosome; N, nucleus. A Gold particles are present along the shaping acrosomal membranes (arrows) of this round spermatid. Bar = 0.17 km. B:
Spectrin is recognized by gold particles all along the shaping head membranes (arrows) of this elongated spermatid. Bar = 0.20 pm. C: Apparently the gold particles distribution (arrows) follows the profile of the acrosome in this spermatid. Bar = 0.12 pm.
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Fig. 17. Rabbit sperm cryosections immunostained for spectrin. Goat anti-rabbit 5 nm has been used as second antibody. IAM, inner acrosomal membrane; OAM, outer acrosomal membrane; PM,plasma membrane. A: Longitudinal section of a sperm head stained with anti-chicken erythrocyte spectrin. Gold particles are placed along the
24 7
head membranes. Bar = 0.22 pm.B: In this cross-section through the four subacrosomal bulges (arrows), stained with anti-a-brain fodrin, gold particles follow the cytoplasmic faces of all the membranes. Bar = 0.20 pm.
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the cortical cytoskeleton plays a role in the spreading of mouse blastomeres (Sobel et al., 1988). The occurrence of a protein belonging to the “spectrin family” in mammalian spermatozoa was hypothesized for the properties of being a major calmodulin binding protein and of being a membrane-skeletal component of many eucharyotic cells. We have analyzed the presence of a spectrin-like protein in rabbit spermiogenesis and spermatozoa (Camatini and Casale, 1988) by SDS electrophoretic separation of sperm proteins and coelectrophoresis of chicken erythrocyte spectrin and sperm extract (Fig. 13) and by the use of anti-spectrin antibodies of different origin (anti-chicken spectrin, anti-brain fodrin, and anti-calf lens p230 spectrin). In immunoblots of sperm extract, the antibodies have recognized a band at the level of the 240 kDa protein subunit (Fig. 14A-C). A bright fluorescence at the acrosomal cap, the postacrosomal region, and the principal piece of the tail (Fig. 15A,B) is evident with the immunofluorescent technique. Moreover immunoelectron microscopy performed both on testis Lowicryl sections (Fig. 16A-C) and sperm cryosections (Fig. 17A,B) show that a spectrin-like protein is recognized by the antibodies used along the head membranes. Anti-spectrin label is on the cytoplasmic face of the PM, the OAM, and along the nuclear face of the IAM. These results suggest that a spectrin-like protein is a component of these membranes, from early (Fig. 16A,C) to mature spermatids (Fig. 16B).
CONCLUDING REMARKS A generalized cytoskeletal model for mammalian spermatozoa does not exist. We should think, rather than of a unique schematic drawing for ejaculated mammalian spermatozoa, of several combinations of different models in different species. It is reasonable to believe that the sperm in its mature form is modulated and maintained by cytoskeletal elements placed in correspondence to membranes, whose shape and function follow a species-specific model. Moreover, capacitation and fertilization events employ molecular mechanisms, which must be differently regulated in sperm belonging to the various species. For example, the migration of guinea pig and rabbit sperm antigens during capacitation is well documented, and epididymal guinea pig sperm do not contain actin (Halenda et al., 1987),while rabbit sperm do (Welch and O’Rand, 1985; Camatini et al., 1987). Several key questions are still unanswered. It is unknown whether the cytoskeletal proteins identified in spermatozoa, identical to these present in other cell systems, play the same functional role as in other cells or whether still unidentified elements are present. It is also unknown if different domains of the spermatozoa possess distinct cytoskeletal assemblies. Another puzzle emerges by the colocalization of some cytoskeletal elements and their regulatory proteins. For example, spectrin-actin, spectrin-calmodulin, and calmodulin-actin associations have been described both in gametes and in early developmental stages of embryos (Virtanen et al., 1984; Reima and Lehtonen, 1985; Camatini and Casale, 1987; Sobel et al., 1988).
Nonetheless, the relationships among such proteins are still unclear in extensively studied cell systems (Pollard, 1984). Evidence against the importance of spectrin-actin interaction is derived by the observation that no change in cell shape or actin distribution appears, when aggregation of spectrin is induced with anti-spectrin antibodies (Mangeat and Burridge, 1984). Moreover, the identification of a-spectrin as the common calmodulin binding subunit of erythroid and nonerythroid spectrin is still unclear (Lazarides and Nelson, 1982). Future research will clarify the molecular regulation of sperm compartmentalization if the efforts that already have obtained numerous encouraging results are focused on the study of the same model in its different aspects from the early stage until the fertilization process. It is perhaps a good suggestion to approach the cytoskeleton of spermatozoa as a system completely different from the well-characterized cytoskeleton of the other cells.
ACKNOWLEDGMENTS The authors are grateful to Dr. Giovanni Bernardini for his valuable suggestions during the preparation of the manuscript. Supported by M.P.I., 60%, 1989, C.N.R. Technology No. 9001981CTll. REFERENCES Bennet, V. (1985)The membrane skeleton of human erythrocytes and its implications for more complex cells. Annu. Biochem., 54:273304. Bloch, R.J., and Morrow, J.S. (1989)An usual p-spectrin associated with clustered acetylcholine receptors. J. Cell Biol., 108:481-493. Camatini, M., Anelli, G., and Casale, A. (1986a)Immunocytochemical localization of calmodulin in intact and acrosome-reacted boar sperm. Eur. J . Cell Biol., 41:89-96. Camatini, M., Anelli, G., and Casale, A. (1986b)Identification of actin in boar spermatids and spermatozoa by immunoelectron microscopy. Eur. J . Cell Biol., 42:311-318. Camatini, M.,and Casale, A. (1987)Actin and calmodulin coexist in the equatorial segment of ejaculated boar sperm. Gamete Res., 17: 97-105. Camatini, M., and Casale, A. (1988)Characterization of cytoskeletal elements in mammalian spermatozoa. In: Sacomeric and Non-Sarcomeric Muscle. U. Carraro, ed. Unipress, Padova, pp. 721-726. Camatini, M., Casale, A., Anelli, G., and Cifarelli, M. (1986~) Immunoelectron microscopic localization of actin in ionophore-treated boar sperm. Cell Biol. Int. Rep., 10231-238. Camatini, M., Casale, A., and Cifarelli, M. (1987)Immunoctyochemical identification of actin in rabbit spermiogenesis and spermatozoa. Eur. J. Cell Biol., 45:274-281. Camatini, M., Colombo, A., Bonfanti, P. (1991)Identification of spectrin and calmodulin in rabbit spermiogenesis and spermatozoa. Mol. Reprod. Dev. 28:62-69. Camatini, M., Franchi, E., and De Curtis, I. (1981)Permeability to lanthanum of blood testis barrier. Anat. Rec., 200:211-224. Casale, A,, Camatini, M., Skalli, O., and Gabbiani, G. (1988)Characterization of actin isoforms in ejaculated boar spermatozoa. Gamete Res., 20133-144. Clarke, G.N., Clarke, F.M., and Wilson, S. (1982)Actin in human spermatozoa. Biol. Reprod., 26:319-327. Clarke, G.N., and Yanagimachi, R. (1978)Actin in mammalian sperm heads. J. Exp. Zool., 205:125-132. Clermont, Y., and Leblond, C.P. (1955)Spermiogenesis in man, monkey, ram and other mammals as shown by periodic acid-Schiff techniques. Am. J . Anat., 96229-253. Damjanov, I., Damjanov, A., Lehto, V.P., and Virtanen, I. (1986)Spectrin in mouse gametogenesis and embryogenesis. Dev. Biol., 114: 132-140. Dym, M., andFawcett, D.W. (1970)The blood-testis in the rat and the
SPERMATOZOA CYTOSKELETAL ELEMENTS physiological compartmentation of the seminiferous epithelium. Biol. Reprod., 3:308-326. Fawcett, D.W. (1975) Ultrastructure and function of the Sertoli cell. In: Handbook of Physiology, Vol. 5. R.O. Greep, ed. Williams and Wilkins, Baltimore, pp. 21-55. Feinberg, J., Weinmann, J., Weinmann, S., Walsh, M.P., Harricane, M.C., Gabrion, J . , and Demaille, J.G. (1981) Immunocytochemical and biochemical evidence for the presence of calmodulin in bull sperm flagellum. Biochim. Biophys. Acta, 673:303-311. Flaherty, S.P., Breed, W.G., and Sarafis, V. (1983) Localization of actin in the sperm head of the plains mouse. J. Exp. Zool., 225: 497-500. Flaherty, S.P., Winfrey, V.P., and Olson, G.E. (1986) Localization of actin in mammalian spermatozoa: A comparison of eight species. Anat. Rec., 216:504-515. Flaherty, S.P., Winfrey, V.P., and Olson. G.E. (1988) Localization of actin-in human, bull, rabbit, and hamster sperm by immunoelectron microscopy. Anat. Rec., 221:599-610. Fouquet, J.P., Kann, M.-L., and Dadoune, J.-P. (1989) Immunogold distribution of actin during spermiogenesis in the rat, hamster, monkey, and human. Anat. Rec., 223:35-42. Gaunt, S.J., Brown, C.R., and Jones, R. (1983) Identification of mobile and fixed antigens on the plasma membrane of rat spermatozoa using monoclonal antibodies. Exp. Cell Res., 144:275-284. Glenney, J.R., Jr., and Glenney, P. (1983) Fodrin is the general spectrin-like protein found in most cells whereas spectrin and the TW protein have a restricted distribution. Cell, 34503-512. Gundersen, G.G., Kalnoski, M.H., and Bulinsky, J.C. (1984) Distinct populations of microtubules: Tyrosinated and nontyrosinated alpha tubulin are distributed differently in vivo. Cell, 38:779-789. Halenda, R.M., Primakoff, P., and Myles, D.G. (1984) Evidence for the presence of actin in guinea pig spermatogenic cells and its loss prior to formation of mature sperm. J. Cell Biol., 99:394a. Halenda, R.M., Primakoff, P., and Myles, D.G. (1987) Actin filaments, localized to the region of the developing acrosome during early stages, are lost during later stages of guinea pig spermiogenesis. Biol. Reprod., 36:491-499. Hopper, J.E., and Kelly, R.B. (1984) Calmodulin is tightly associated with synaptic vesicles independent of calcium. J . Biol. Chem., 259: 148-153. Jones, H.P., Bradford, M.M., McRorie, R.A., and Cormier, M.J. (1978) High levels of calcium-dependent modulator protein in spermatozoa and its similarity to brain modulator protein. Biochem. Biophys. Res. Commun., 82:1264-1272. Jones, H.P., Lenz, R.W., Palevitz, B.A., Cormier, M.J. (1980) Calmodulin localization in mammalian spermatozoa. Proc. Natl. Acad. Sci. U.S.A., 77:2772-2776. Korn, E.D. (1982) Actin polymerizaiton and its regulation by proteins from nonmuscle cells. Physiol. Rev., 62:672-737. Lakoski, K., Williams, C., and Saling, P. (1989) Proteins of the acrosomal region in mouse sperm: Immunological probes reveal posttesticular modifications. Gamete Res., 23:21-37. Lazarides, E., and Nelson, W.J. (1982) Expression of spectrin in nonerythroid cells. Cell, 31505-508. Longo, F.J., Krone, G., and Franke, W.W. (1987) Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: A novel class of cytoskeletal elements. J . Cell Biol., 105:1105-1120. Mangeat, P.H., and Burridge, K. (1984) Immunoprecipitation of nonerythrocyte spectrin within live cells following microinjection of spectrin antibodies: Relation to cytoskeletal structures. J . Cell Biol., 981363-1377. Marchesi, V.T. (1985) Stabilizing infrastructure of cell membranes. Annu. Rev. Cell Biol., 1:531-562. Masri, B.A., Russel, L.D., and Vogl, A.W. (1987) Distribution of actin in spermatids and adjacent Sertoli cell regions of the rat. Anat. Rec., 218:20-26. Mercier, F., Reggio, H., Devilliers, G., Bataille, D., and Mangeat, P. (1989) Membrane-cytoskeleton dynamics in rat parietal cells: Mobilization of actin and spectrin upon stimulation of gastric acid secretion. J. Cell Biol., 108:441-453. Miller, L.M., Kalnoski, M., Yunossi, Z., Bulinski, J.C., and Reisler, E. (1987) Antibodies derected against N-terminal residues on actin do not block acto-myosin binding. Biochemistry, 26:6064-6070. Morrow, M.S. (1989) The spectrin membrane skeleton: Emerging concepts. Curr. Opin. Cell Biol., 1:23-29. Myles, D.G., F’rimakoff, P., and Bellv6, A.R. (1981) Surface domains of the guinea pig sperm defined with monoclonal antibodies. Cell, 23: 433-439.
249
Noland, T.D., Van Eldik, L.J., Garbers, D.L., and Burgess, W.H. (1985) Distribution of calmodulin and calmodulin-binding proteins in membranes from bovine epididymal spermatozoa. Gamete Res., 11:297-303. Ochs, D., and Wolf, D.P. (1985) Actin in ejaculated human sperm cell. Biol. Reprod., 33:1223-1226. Ochs, D., Wolf, D.P., and Ochs, R.L. (1986) Intermediate filament proteins in human sperm heads. Exp. Cell Res., 167:495-504. Olson, G.E., Winfrey, V.P., and Flaherty, S.P. (1987) Cytoskeletal assemblies of mammalian spermatozoa. Ann. N.Y. Acad. Sci., 29: 222-246. Olson, G.E., Winfrey, V.P., Garbers, D.L., and Noland, T.D. (1985) Isolation and characterization of a macromolecolar complex associated with the outer acrosomal membrane of bovine spermatozoa. Biol. Reprod., 33:761-779. O’Rand, M., and Fisher, S. (1987) Localization of zona pellucida binding sites on rabbit spermatozoa and induction of the acrosome reaction by solubilized zonae. Dev. Biol., 119551-559. O’Rand, M.G., Matthews, J.E., Weech, J.E., and Fisher, S.J. (1985) Identification of mna binding proteins of rabbit, pig, human, and mouse spermatozoa on nitrocellulose blots. J. Exp. Zool., 235423428. Otey, C.A., Kalnosky, M.H., and Bulinski, J.C. (1986)A procedure for the immunoblotting of proteins separated on isoelectric focusing gels. Anal. Biochem., 157:71-76. Pelletier, R.M. (1988) Cyclic modulation of Sertoli cell junctional complexes in a seasonal breeder: The mink (Mustela vision). Am. J . Anat., 183:68-102. Peterson, R.N., Bozzola, J.J., Hunt, W.P., and Darabi, A. (1990) Characterization of membrane-associated actin in boar spermatozoa. J. Exp. Zool., 253:202-204. Peterson, R.N., Chaundhry, P., and Tibbs, B. (1989) Calcium-binding protein of boar spermatozoan plasma membranes: Identification and partial characterization. Gamete Res., 23:49-60. Phillips, M.D. (1972) Substructure of the mammalian acrosome. J. Ultrastruct. Res., 38:591-604. Pollard, T.D. (1984) Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins. J. Cell Biol., 99:19701980. Pollard, T.D., and Cooper, J.A. (1986) Actin and actin-binding proteins. A critical evolution of mechanism and functions. Ann. Rev. Biochem., 55:987-1035. Primakoff, P., and Myles, D.G. (1983) A map of the guinea pig sperm surface constructed with monoclonal antibodies. Dev. Biol., 98:417428. Reima, I., and Lehtonen, E. (1985) Localization of nonerytroid spectrin and actin in mouse oocytes and preimplantation embryos. Differentiation, 30:68-75. Russel. L.D.. Goh. J.C. Rashed. R.M.A.. and Vod. A.W. (1988) The consequences of actin disruption a t Sertoli ec6plasmic specialization sites facing spermatids after in vivo exposure of rat testis to cytochalasin D. Biol. Reprod., 39:105-118. Russel, L.D., and Peterson, R.N. (1985) Sertoli cell junction: Morphological and functional correlates. Int. J . Cyt., 94:177-211. Russel, L.D., Weber, J.E., and Vogl, A.W. (1986) Characterization of filaments within the subacrosomal space of rat spermatids during spermiogenesis. Tissue Cell, 183387-898. Schatten, H., Cheney, R., Balczon, R., Cline, C., Simerly, C., and Schatten, G. (1986) Localization of fodrin during fertilization and early development of sea urchin and mice. Dev. Biol., 118:457-466. Sobel, J.S., Goldstein, E.G., Venuti, J.M., and Welsh, J.M. (1988) Spectrin and calmodulin in spreading mouse blastomeres. Dev. Biol., 126:47-56. Talbot, P., and Kleve, M.G. (1978) Hamster sperm cross react with anti-actin. J. Exp. Zool., 204:131-136. Tamblyn, T.M. (1980) Identification of actin in boar epididymal spermatozoa. Biochem. Biophys. Res. Commun., 62:328-335. Van Eldik, L.J., and Burgess, W.H. (1983) Analytical subcellular distribution of calmodulin and calmodulin-binding proteins in normal and virus-transformed fibroblasts. J. Biol. Chem., 258:4539-4547. Virtanen, I., Badley, R.A., Paasivuo, R., and Lehto, V.P. (1984) Distinct cytoskeletal domains revealed in sperm cells. J. Cell Biol., 99:1083-1091. Vogl, A.W. (1988) Changes in the distribution of microtubules in rat Sertoli cells during spermagogenesis. Anat. Rec., 222:34-41. Vogl, A.W. (1989) Distribution and function of organized concentra-
250
M. CAMATINI ET AL.
tions of actin filaments in mammalian spermatogenic cells and Sertoli cells. Int. Rev. Cyt., 119:l-56. Vogl, A.W., Grove, B.D., and Lew, G.J.(1986) Distribution of actin in Sertoli cell ectoplasmic specializations and associated spermatids in the ground squirrel testis. Anat. Rec., 215331-341. Weinman, S., Ores-Carton, C., Escaig, F., Feinberg, J., and Puszkin,
S. (1986) Calmodulin immunoelectron microscopy: Redistribution during ram spermatogenesis and epididymal maturation. II. J. Histochem. Cytochem., 32:1181-1193. Welch, J.A., and O’Rand, M.G. (1985) Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit. Dev. Biol., 109:411-417.
